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Prognostic impact of preoperative monocyte counts in patients with resected lung adenocarcinoma
Lung Cancer 85 (2014) 457–464
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Prognostic impact of preoperative monocyte counts in patients with resected lung adenocarcinoma Shogo Kumagai a , Satoshi Marumo a , Tsuyoshi Shoji a , Minoru Sakuramoto a , Tatsuya Hirai a , Takafumi Nishimura b , Nobuyoshi Arima c , Motonari Fukui a , Cheng-long Huang a,∗ a
Respiratory Disease Center, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan Department of Clinical Oncology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan c Department of Hematology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan b
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 19 June 2014 Accepted 21 June 2014 Keywords: Lung adenocarcinoma Peripheral monocyte counts Overall survival Recurrence-free survival Curative resection Prognostic factor
a b s t r a c t Objectives: Increasing evidence suggests that an elevated peripheral monocyte count at presentation predicts a poor prognosis in various types of malignancy, including malignant lymphoma. In lung adenocarcinoma, tumor-associated macrophages (TAMs) were reported to be associated with a poor prognosis. However, it is unknown if an elevated peripheral monocyte count is associated with a poor prognosis in lung adenocarcinoma. This study assessed the prognostic impact of the preoperative peripheral monocyte count in lung adenocarcinoma. Materials and methods: We retrospectively analyzed 302 consecutive patients with lung adenocarcinoma who received curative resection at Kitano Hospital. The receiver operating characteristic (ROC) curve for the peripheral monocyte count was used to determine the cut-off value. The relations between peripheral monocyte counts and clinicopathological factors were assessed. We also evaluated the impacts of possible prognostic factors including the preoperative peripheral monocyte count on survival, using the two-tailed log-rank test and Cox proportional hazards model. In addition, immunohistochemical staining for CD68 was performed to evaluate the monocytes in primary tumors. Results: A peripheral monocyte count of 430 mm−3 was the optimal cut-off value for prognosis. An elevated peripheral monocyte count was significantly associated with sex, performance status, smoking history, chronic obstructive pulmonary disease and interstitial lung disease. The two-tailed log-rank test demonstrated that patients with an elevated peripheral monocyte count experienced a poorer recurrence-free survival (RFS) and overall survival (OS) (P = 0.0063, P < 0.0001, respectively). In the multivariate analysis an elevated peripheral monocyte count was shown to be an independent prognostic factor for the RFS and OS (HR: 1.765; 95% CI: 1.071–2.910; P = 0.0258, HR: 4.339; 95% CI: 2.032–9.263; P = 0.0001, respectively). Furthermore, numbers of the monocytes in primary tumors significantly correlated with peripheral monocyte counts (r = 0.627, P < 0.0001). Conclusion: The preoperative peripheral monocyte count is an important prognostic factor for patients with lung adenocarcinoma after curative resection. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is one of the most common causes of cancerrelated death and adenocarcinoma is the most popular type of
∗ Corresponding author at: Respiratory Disease Center, Tazuke Kofukai Medical Research Institute, Kitano Hospital, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan. Tel.: +81 6 6312 8831; fax: +81 6 6361 8867. E-mail address: [email protected] (C.-l. Huang). http://dx.doi.org/10.1016/j.lungcan.2014.06.015 0169-5002/© 2014 Elsevier Ireland Ltd. All rights reserved.
lung cancer, accounting for approximately one-half of lung cancer cases. In lung adenocarcinoma, tumor-associated macrophages (TAMs) were reported to be associated with a poor prognosis [1,2]. TAMs are derived from monocytes and myeloid progenitor cells [3]. An elevated peripheral monocyte count at presentation has recently been shown to predict poor prognosis in various types of malignancy, including malignant lymphomas [4–7]. However, it is unknown if an elevated peripheral monocyte count is associated with a poor prognosis in lung adenocarcinoma. This study assessed the prognostic impact of the preoperative peripheral monocyte
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count on the recurrence-free survival (RFS) and overall survival (OS) in patients with lung adenocarcinoma who received curative resection. 2. Materials and methods
compared using the chi-square test. Continuous variables were compared using the Wilcoxon rank-sum test. All statistical analyses were performed using the statistical software R version 2.13.1 (R Foundation for statistical computing, Vienna, Austria). All P values are 2-sided, and P values less than 0.05 were considered statistically significant.
2.1. Patients We conducted a retrospective analysis of patients diagnosed with lung adenocarcinoma who underwent surgery at the Tazuke Kofukai Medical Research Institute, Kitano Hospital between January 2007 and December 2012. All patients met the following criteria: pathological confirmation of lung adenocarcinoma; complete curative resection; no preoperative treatment; no microscopic residual tumor; no history of transplantation, immunosuppression; no evidence of infection such as pneumonia before surgery; no treatment for concomitant autoimmune diseases with immunosuppressive therapy; no history of acute exacerbation of chronic obstructive pulmonary disease (COPD) or interstitial lung disease (ILD) within a month before surgery; negative for anti-HIV; and availability of laboratory data and follow-up information. COPD was diagnosed on the basis of the Global Initiative for Chronic Obstructive Lung Disease [8]. ILD was diagnosed by two pulmologists, based on medical history, physical examination and abnormalities compatible with bilateral lung fibrosis on high-resolution computed tomography. A peripheral venous blood sample was collected from each patient within a month before surgery. A blood test was performed using a fully automated blood cell counting system. Histological classification was carried out according to the WHO guidelines [9]. Disease stages were based on the 7th edition of TNM classification of malignant tumors [10]. Study approval was granted by the ethical committee of the Tazuke Kofukai Medical Research Institute, Kitano Hospital, in accordance with the Declaration of Helsinki. 2.2. Evaluation of clinicopathological factors The following clinical characteristics were retrieved from the available clinical records: age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, smoking history, pathological stage, pathological tumor status, pathological lymph node status, pleural invasion, vascular invasion, lymphatic permeation, peripheral leukocyte counts, peripheral monocyte counts, peripheral neutrophil counts, peripheral lymphocyte counts, and C-reactive protein (CRP) levels. RFS was measured from the date of surgery until the date of recurrence or until the date the patient was last known to be disease free. OS was measured from the date of surgery until the date of death from any cause or until the date on which the patient was last known to be alive. 2.3. Statistical analysis We estimated OS and RFS using the Kaplan–Meier analysis [11]. Differences between survival curves were tested for statistical significance using the two-tailed log-rank test. Univariate and multivariate prognostic analyses were performed for OS and RFS outcomes using the Cox proportional hazards model. For multivariate survival analysis, a stepwise backward procedure to derive a final model of the variables that had a significant independent relationship with survival was employed. To remove a variable from the model, the corresponding P value had to be >0.10. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off values for peripheral leukocyte counts, peripheral neutrophil counts, peripheral monocyte counts, and peripheral lymphocyte counts; values with maximum joint sensitivity and specificity were selected. Categorical variables were
2.4. Immunohistochemistry Formalin-fixed paraffin-embedded tissue was cut into 4m sections and mounted on poly-l-lysine-coated slides. After deparaffinization and rehydration, the slides were heated in a microwave for 30 min in a 10-mol/L citrate buffer solution at pH 6.0, and were cooled to room temperature for 20 min. After quenching the endogenous peroxidase activity with 0.3% H2 O2 (in absolute methanol) for 30 min, the sections were treated with 5% bovine serum albumin to block non-specific staining. Individual sections were incubated overnight with mouse monoclonal antihuman CD68 antibody (Clone KP1, Dako, Japan) at a 1:100 dilution. The slides were then incubated with EnVision (Dako, Denmark) for 1 h at room temperature. Then, the antibody binding was visualized with 3,3 -diaminobenzidine tetrahydrochloride. Finally, the sections were lightly counterstained with Mayer’s hematoxylin. All of the immunostained sections were reviewed by two authors (S.K. and C.H.) without knowledge of the patients’ characteristics. CD68positive round cells in the stroma of the cancer tissue were counted as macrophage. Cases with discrepancies were jointly reevaluated until a consensus was reached. 3. Results 3.1. Patients Data from 445 patients diagnosed with non-small cell lung cancer (NSCLC) who underwent surgery at our hospital between January 2007 and December 2012 were obtained from the hospital’s database. One hundred and forty-three patients were excluded due to diagnosis of histology other than lung adenocarcinoma (N = 107), preoperative treatment (N = 14), and incomplete resection (N = 22). Thus 302 patients were included in this study. The clinicopathological characteristics of patients are shown in Table 1. There were 136 men and 166 women. The median age at the time of surgery was 67 years (range: 34–87 years). The median follow-up duration was 33.4 months (range: 1.0–77.4 months). Fifty-seven patients received postoperative adjuvant chemotherapy and 72 patients experienced recurrences of lung adenocarcinoma. The 5-year recurrence-free survival rate and overall survival rate of all 302 patients were 70.2% (pathological stage I, 83.7%; pathological stage II, 43.4%; pathological stage IIIA, 34.9%) and 84.0% (pathological stage I, 87.5%; pathological stage II, 73.7%; pathological stage IIIA, 75.5%), respectively. 3.2. Optimal cut-off values for leukocyte, neutrophil, monocyte and lymphocyte counts ROC curves for peripheral leukocyte counts, peripheral neutrophil counts, peripheral monocyte counts and peripheral lymphocyte counts were used to determine cut-off values. The area under the curve (AUC) for peripheral leukocyte counts was 0.560 (95% confidence interval (95% CI), 0.443–0.677). A leukocyte count of 5400 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (88.9% sensitivity and 47.0% specificity). The AUC for peripheral neutrophil counts was 0.606 (95% CI: 0.467–0.744). A neutrophil count of 2900 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve
S. Kumagai et al. / Lung Cancer 85 (2014) 457–464 Table 1 Clinicopathological characteristics of patients. Variables
Total (N = 302)
Age (median), years Sex (male/female) ECOG performance status (0/1/2) Smoking history (never/ever) Comorbidity and past history COPD ILD Old pulmonary tuberculosis Bronchial asthma Hypertension Diabetes mellitus Hyperlipidemia Ischemic heart disease Arrhythmia Resected side (right/left) Surgical procedure (Lob/Seg/Wedge) Pathological stage (I/II/IIIA) Tumor status (T1/T2/T3/T4) Lymph node status (N0/N1/N2) Pleural invasion (present/absent) Vascular invasion (present/absent) Lymphatic permeation (present/absent) Leukocyte counts (mean ± SD), mm−3 Neutrophil counts (mean ± SD), mm−3 Monocyte counts (mean ± SD), mm−3 Lymphocyte counts (mean ± SD), mm−3 C-reactive protein (mean ± SD), mg/dL Postoperative adjuvant chemotherapy Recurrences of lung adenocarcinoma Cause of death (lung cancer/others/unknown)
67 (34–87) 136/166 271/30/1 159/143 38 22 15 22 84 29 40 19 20 180/122 259/39/4 220/39/43 173/97/20/12 254/14/34 71/231 41/260 23/276 5900 ± 1700 3600 ± 1600 410 ± 150 1700 ± 560 0.28 ± 0.52 57 72 15/16/4
Abbreviations: SD, standard deviation; ECOG, Eastern Cooperative Oncology Group; COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease; Lob, lobectomy or bilobectomy; Seg, segmentectomy; Wedge, wedge resection.
(100.0% sensitivity and 42.6% specificity). The AUC for peripheral monocyte counts was 0.711 (95% confidence interval (CI), 0.622–0.800) (Suppl. 1). A peripheral monocyte count of 430 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (69.7% sensitivity and 66.9% specificity). The AUC for peripheral lymphocyte counts was 0.663 (95% CI: 0.444–0.882). A lymphocyte count of 1400 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (66.7% sensitivity and 70.4% specificity). Supplementary Fig. 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan.2014.06.015. 3.3. The relations between peripheral monocyte counts and clinicopathological factors The relations between the peripheral monocyte count and clinicopathological factors are shown in Table 2. An elevated peripheral monocyte count was significantly associated with sex, ECOG performance status, smoking history, COPD, and ILD. An elevated peripheral monocyte count was also significantly associated with recurrences of lung adenocarcinoma. 3.4. Evaluation of the prognostic impact of peripheral monocyte counts The Kaplan–Meier analysis was performed to determine if the peripheral monocyte count was associated with the RFS and OS. The RFS and OS were significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 , in all stages (P = 0.0063 and P < 0.0001, respectively) (Fig. 1A and B). In the pathological stage I patients, the OS and RFS were significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the
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group with a peripheral monocyte count ≤430 mm−3 (P = 0.0050 and P = 0.0149, respectively) (Fig. 1C and D). In the pathological stage II patients, the RFS was not significantly shorter in the group with a peripheral monocyte count >430 mm−3 (P = 0.0549) (Fig. 1E). However, the OS was significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 (P = 0.0099) (Fig. 1F). In the pathological stage IIIA patients, there was no significant difference in RFS between the two groups (P = 0.1740) (Fig. 1G). In contrast, the OS was significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 (P = 0.0398) (Fig. 1H). 3.5. Univariate and multivariate analyses of factors associated with prognosis As for the RFS, the univariate analysis identified eleven significant risk factors: sex, ECOG performance status, tumor status, lymph node status, pleural invasion, vascular invasion, lymphatic permeation, a high peripheral monocyte counts, a low peripheral lymphocyte count, a high CRP level, and ILD (Table 3). In the multivariate analysis, a high peripheral monocyte count was shown to be a statistically significant independent predictor of the RFS (HR: 1.765; 95% CI: 1.071–2.910; P = 0.0258). The other independent prognostic factors were sex (HR: 1.689; 95% CI: 1.023–2.789, P = 0.0406), tumor status (HR: 2.472; 95% CI: 1.458–4.193, P = 0.0008), lymph node status (HR: 3.400; 95% CI: 2.044–5.657, P < 0.0001), and a low lymphocyte count (HR: 2.128; 95% CI: 1.319–3.433, P = 0.0020), and ILD (HR: 2.093, 95% CI: 1.063–4.121, P = 0.0327). The univariate analysis showed ten significant risk factors for the OS: age, sex, ECOG performance status, tumor status, a high peripheral neutrophil count, a high peripheral monocyte count, a low peripheral lymphocyte count, a high CRP level, COPD, and ILD (Table 4). The multivariate analysis revealed that a high peripheral monocyte count was a statistically significant independent prognostic factor of the OS (hazard ratio (HR): 4.339; 95% CI: 2.032–9.263; P = 0.0001). The other independent prognostic factors were tumor status (HR: 2.131; CI: 1.026–4.425, P = 0.0425), a low peripheral lymphocyte count (HR: 3.365; 95% CI: 1.683–6.725, P = 0.0006), and ILD (HR: 3.041; 95% CI: 1.337–6.914, P = 0.0080). The peripheral monocyte count was analyzed in an attempt to further discriminate clinical outcomes in lung adenocarcinoma by other prognostic factors: pathological stage; tumor status; lymph node status; peripheral leukocyte counts; peripheral neutrophil counts; peripheral lymphocyte counts; CRP. Interactions of a peripheral monocyte count of >430 mm−3 vs. a peripheral monocyte count of ≤430 mm−3 were shown in a Forest plot (Fig. 2). Patients with a peripheral monocyte count of >430 mm−3 showed a poorer OS, compared to those with a peripheral monocyte count of ≤430 mm−3 in 13 of 14 subgroups, the exception being the group of patients with T1. 3.6. Correlations between numbers of monocytes in primary tumors and peripheral monocyte counts In order to evaluate the monocytes in the stroma of the primary tumor, immunohistochemistry was performed using consecutive 71 patients who underwent surgery at our hospital between June 2011 and December 2012 (Suppl. 2). The mean number of CD68-positive monocytes in the stroma of primary tumors was 29.7 ± 29.9 (median: 20, range: 0–122). The numbers of monocytes in the stroma of primary tumors significantly correlated
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Fig. 1. The Kaplan–Meier analyses of recurrence-free survival and overall survival stratified by peripheral monocyte counts (<430 vs. ≥430 mm−3 ) are shown for all pathological stages (A, B, respectively), for pathological stage I (C, D, respectively), for pathological stage II (E, F, respectively), and for pathological stage IIIA (G, H, respectively).
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Table 2 Relationship between preoperative peripheral monocyte counts and clinicopathological factors. Monocyte counts
Variables
Age (median), years Sex (male/female) ECOG performance status (0/1/2) Smoking history (never/ever) Comorbidity and past history COPD ILD Old pulmonary tuberculosis Bronchial asthma Hypertension Diabetes mellitus Hyperlipidemia Ischemic heart disease Arrhythmia Resected side (right/left) Surgical procedure (Lob/Seg/Wedge) Pathological stage (I/II/IIIA) Tumor status (T1/T2/T3/T4) Lymph node status (N0/N1/N2) Pleural invasion (present/absent) Vascular invasion (present/absent) Lymphatic permeation (present/absent) Postoperative adjuvant chemotherapy Recurrences of lung adenocarcinoma Cause of death (lung cancer/others/unknown)
P-value
≤430 mm−3 N = 192
>430 mm−3 N = 110
68 (34–87) 66/126 179/13/0 120/72
68.5 (45–87) 70/40 92/17/1 39/71
0.7977 <0.0001 0.0115 <0.0001
13 8 9 14 48 18 25 11 11 114/78 169/21/2 139/27/26 110/60/15/7 163/8/21 42/150 23/169 13/179 37 36 4/6/1
24 14 6 8 36 11 15 8 9 66/44 90/18/2 81/12/17 63/37/5/5 91/6/13 29/81 18/92 10/100 20 36 11/10/3
0.0002 0.0099 0.7870 0.9999 0.1820 0.8420 0.8620 0.6270 0.4730 0.9999 0.3420 0.7050 0.7130 0.8190 0.3990 0.2990 0.5030 0.8790 0.0076 0.8740
Abbreviations: SD, standard deviation; ECOG, Eastern Cooperative Oncology Group; COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease; Lob, lobectomy or bilobectomy; Seg, segmentectomy; Wedge, wedge resection.
with peripheral monocyte counts (r = 0.627, P < 0.0001). The median number of monocytes was used to divide the 71 cases into two groups: monocyte-high; above the median value, monocyte-low; below the median value. The peripheral monocyte counts were 470 ± 146 mm−3 in monocyte-high tumors, and 328 ± 117 mm−3 in monocyte-low tumors. The peripheral monocyte counts were significantly higher in monocyte-high tumors than in monocyte-low tumors (P < 0.0001).
Supplementary Fig. 2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan.2014.06.015. 4. Discussion We demonstrated that a high peripheral monocyte count was a poor prognostic factor in lung adenocarcinoma. Monocytosis at presentation was reported to predict a poor prognosis
Table 3 Prognostic impacts on recurrence-free survival in lung adenocarcinoma. Variables Univariate analysis Age Sex ECOG performance status Smoking history Tumor status Lymph node status Pleural invasion Vascular invasion Lymphatic permeation Leukocyte counts Monocyte counts Neutrophil counts Lymphocyte counts CRP COPD ILD Multivariate analysis Sex Tumor status Lymph node status Monocyte counts Lymphocyte counts ILD
HR
95% CI
P-value
≥70 Male 1/2 Ever T2/T3/T4 N1/N2 Present Present Present >5400 mm−3 >430 mm−3 >2900 mm−3 ≤1400 mm−3 >0.1 mg/dL Present Present
1.499 1.876 1.924 0.652 3.502 5.081 3.370 3.353 4.332 1.377 1.886 1.436 1.762 1.885 1.616 3.507
0.911–2.304 1.175–2.995 1.009–3.668 0.409–1.038 2.130–5.756 3.156–8.178 2.120–5.356 1.909–5.887 2.336–8.033 0.858–2.211 1.187–2.995 0.879–2.346 1.108–2.803 1.186–2.995 0.868–3.010 1.878–6.547
0.1174 0.0084 0.0469 0.0716 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.1850 0.0072 0.1489 0.0168 0.0073 0.1301 <0.0001
Male T2/T3/T4 N1/N2 >430 mm−3 ≤1400 mm−3 Present
1.689 2.472 3.400 1.765 2.128 2.093
1.023–2.789 1.458–4.193 2.044–5.657 1.071–2.910 1.319–3.433 1.063–4.121
0.0406 0.0008 <0.0001 0.0258 0.0020 0.0327
Abbreviations: ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; 95% CI, 95% confidence interval; CRP, C-reactive protein; COPD, chronic obstructive lung disease; ILD, interstitial lung disease.
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Table 4 Prognostic impacts on overall survival in lung adenocarcinoma. Variables Univariate analysis Age Sex ECOG performance status Smoking history Tumor status Lymph node status Pleural invasion Vascular invasion Lymphatic permeation Leukocyte counts Neutrophil counts Monocyte counts Lymphocyte counts CRP COPD ILD Multivariate analysis Tumor status Monocyte counts Lymphocyte counts ILD
HR
95% CI
P-value
≥70 Male 1/2 Ever T2/T3/T4 N1/N2 Present Present Present >5400 mm−3 >2900 mm−3 >430 mm−3 ≤1400 mm−3 >0.1 mg/dL Present Present
2.856 2.471 2.610 0.607 2.428 1.913 1.717 2.072 1.376 1.483 2.337 3.848 2.429 2.166 2.368 5.717
1.436–5.677 1.227–4.976 1.131–6.024 0.311–1.187 1.217–4.842 0.895–4.092 0.859–3.430 0.850–5.053 0.420–4.507 0.743–2.959 1.059–5.159 1.884–7.858 1.246–4.737 1.107–4.237 1.068–5.252 2.662–12.28
0.0028 0.0113 0.0246 0.1447 0.0118 0.0943 0.1261 0.1093 0.5978 0.2633 0.0356 0.0002 0.0092 0.0240 0.0339 <0.0001
T2/T3/T4 >430 mm−3 ≤1400 mm−3 Present
2.131 4.339 3.365 3.041
1.026–4.425 2.032–9.263 1.683–6.725 1.337–6.914
0.0425 0.0001 0.0006 0.0080
Abbreviations: ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; 95% CI, 95% confidence interval; CRP, C-reactive protein; COPD, Chronic obstructive lung disease; ILD, interstitial lung disease.
in classical Hodgkin lymphoma [4], nodular lymphocytepredominant Hodgkin lymphoma [5], follicular lymphoma [6], and mantle cell lymphoma [7]. To the best of our knowledge, this is the first report showing the prognostic significance of the peripheral monocyte count in lung adenocarcinoma, one of the most popular solid tumors.
Peripheral monocytes and myeloid progenitor cells grow into TAMs when entering tumors [3]. TAMs are classified into the two phenotypes: the classically activated type 1 macrophages (M1) and the alternatively activated type 2 macrophages (M2) [12]. Macrophages are polarized along the M1 pathway, affected by bacterial moieties such as lipopolysaccharide (LPS), Th1
Fig. 2. Subgroup analysis of overall survival for peripheral monocyte counts (<430 vs. ≥430/mm−3 ) is shown in the Forest plot.
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cytokine interferon-gamma (IFN␥). Activated M1 macrophages, which are adapted to promote an anti-tumor response, eliminate tumor cells, present antigen to T cells for an adaptive immune response, and produce cytokines [12,13]. In contrast, exposure to Th2 and tumor-derived cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), transforming growth factor beta (TGF-) or prostaglandin E2 (PGE2) promote M2 polarization [14]. M2 macrophages, suitable for tumor development, express high levels of class A scavenger receptor (CD204) and mannose receptor (CD163) [15,16], suppress Th1 mediated-inflammation through IL-10 and IL-1b production, and promote angiogenesis via vascular endothelial growth factor (VEGF) production [17–19]. It was shown that the infiltration of macrophages in tumor islets was associated with a good prognosis in contrast to infiltration in the tumor stroma, related to a worse prognosis in NSCLC [20,21]. These findings suggest that the localization of TAMs plays an important role in predicting prognosis. Recent studies have shown that macrophages in the tumor islets which are associated with a good prognosis are mainly of the M1 phenotype [22,23]. On the other hand, TAMs of the M2 phenotype, which are located in the tumor stroma, were correlated with lymph node metastasis and poor prognosis in lung adenocarcinoma [1,2,24]. In classical Hodgkin lymphoma, the correlation between the percentage of peripheral monocytes at diagnosis and number of CD163+ TAMs in lesional tissue was reported [25]. In the present study, an elevated peripheral monocyte count is significantly associated with a poor prognosis in lung adenocarcinoma. Furthermore, numbers of monocytes in primary tumors significantly correlated with peripheral monocyte counts. A recent study has also reported that circulating macrophages predict postoperative recurrence in non-small-cell lung cancer patients [26]. These results may suggest a complex relation between peripheral monocytes and TAMs of the M2 immunosuppressive phenotype in lung adenocarcinoma. In this study, an elevated peripheral monocyte count was significantly associated with sex, ECOG performance status, smoking history, COPD and ILD. Cigarette smoking was reported to increase the peripheral monocyte count [27]. Systemic inflammation is associated with impaired lung function, particularly among those with extensive cigarette smoking [28]. The proportion of smokers in men is known to be larger than that in women. In addition, COPD was shown to be associated with lung cancer mortality [29]. On the other hand, in patients with ILD the peripheral monocyte count is elevated [30]. Recently, Voltolini et al. reported that long-term survival of patients with NSCLC and ILD is significantly lower when compared with patients without ILD [31]. In the present study, the univariate analysis showed that both COPD and ILD were significantly associated with the OS of lung adenocarcinoma. Besides, the multivariate analysis demonstrated that ILD was a significant prognostic factor of the OS and RFS, and that the peripheral monocyte count predicted the prognosis of the OS and RFS, independent of ILD. We also evaluated the prognostic importance of the peripheral monocyte count in patients without COPD or ILD (N = 251). The univariate analysis demonstrated that a high peripheral monocyte count was a statistically significant predictor of the OS (P = 0.0087). The result reinforced the hypothesis that the peripheral monocyte count is associated with the prognosis of lung adenocarcinoma, independent of COPD and ILD. Both peripheral neutrophil counts [32,33] and CRP levels [34] have been shown to be independent prognostic factors in NSCLC. In this study, however, we showed that an elevated peripheral neutrophil count and CRP level were poor prognostic factors only in the univariate analysis, not in the multivariate analyses. Both peripheral neutrophil counts and CRP levels were supposed to reflect
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the nature and severity of inflammation associated with tumor. In contrast, an elevated peripheral monocyte count was shown to predict a poor RFS and OS in the multivariate analysis, as well as in the univariate analysis. In addition, as shown in the Forest plot, the peripheral monocyte count was found to predict the prognosis of lung adenocarcinoma in 13 subgroups. These findings may suggest that peripheral monocyte counts is a very strong prognostic factor and play a significant role in the tumor progression. The preoperative peripheral lymphocyte count was reported to be an important prognostic factor in node-negative NSCLC [35]. The present study revealed that the peripheral lymphocyte count was a prognostic factor in patients with lung adenocarcinoma including node-positive carcinoma that received complete resection. In addition, the multivariate analysis indicated that a low peripheral lymphocyte count predicted a poor RFS and OS, independent of other possible prognostic factors. In previous study, the cut-off values of the peripheral lymphocyte count differed from the cut-off value found in the present study, possibly reflecting the difference in the type of histology. Fifty-seven patients received postoperative adjuvant chemotherapy in this study. We evaluated the impact of postoperative adjuvant chemotherapy on the OS. However, there was no difference in the OS between patients with postoperative adjuvant chemotherapy and those without by the two-tail log-rank test (P = 0.2330). Among 245 patients without postoperative adjuvant chemotherapy, the two-tail log-rank test showed that peripheral monocyte count >430 mm−3 was also a significant prognostic factor for the OS in all pathological stages (N = 245), pathological stage I (N = 213), and pathological stage II–IIIA (N = 32) (OS, P = 0.0008, P = 0.0252, P = 0.0057, respectively; RFS, P = 0.0003, P = 0.0039, P = 0.0018, respectively). A peripheral monocyte count >430 mm−3 was shown to be associated with a poor prognosis, even negating the effects of postoperative adjuvant chemotherapy. In the present study, twenty patients (57.1% of all cases of death) have died of causes unrelated to lung adenocarcinoma. Therefore, we also assessed the cancer-specific survival (CSS), which was measured from the date of surgery until the date of death from lung adenocarcinoma or its treatment, with data for patients who died of unrelated causes censored at the time of death or last follow-up. The univariate analysis demonstrated that a peripheral monocyte count >430 mm−3 was a significant predictor of a poor CSS (P = 0.0074). This result may support the hypothesis that peripheral monocytes are significantly associated with the progression of lung adenocarcinoma. In conclusion, our study demonstrated that preoperative peripheral monocyte count is a useful prognostic marker in lung adenocarcinoma, supporting the notion that the TAMs have a prognostic impact on clinical outcome in lung adenocarcinoma. The peripheral monocyte count, obtained from a CBC before surgery, may be one of the simplest measures as a prognostic marker in lung adenocarcinoma. Limitations of this study include retrospective analysis, relatively short follow-up duration. Further prospective studies are required to confirm the present findings and to clarify the mechanism in which peripheral monocytes have impacts on clinical outcomes.
Conflict of interest statement All authors have no potential conflicts of interest to report.
Acknowledgements No financial support for this study was provided.
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Prognostic impact of preoperative monocyte counts in patients with resected lung adenocarcinoma Shogo Kumagai a , Satoshi Marumo a , Tsuyoshi Shoji a , Minoru Sakuramoto a , Tatsuya Hirai a , Takafumi Nishimura b , Nobuyoshi Arima c , Motonari Fukui a , Cheng-long Huang a,∗ a
Respiratory Disease Center, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan Department of Clinical Oncology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan c Department of Hematology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan b
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 19 June 2014 Accepted 21 June 2014 Keywords: Lung adenocarcinoma Peripheral monocyte counts Overall survival Recurrence-free survival Curative resection Prognostic factor
a b s t r a c t Objectives: Increasing evidence suggests that an elevated peripheral monocyte count at presentation predicts a poor prognosis in various types of malignancy, including malignant lymphoma. In lung adenocarcinoma, tumor-associated macrophages (TAMs) were reported to be associated with a poor prognosis. However, it is unknown if an elevated peripheral monocyte count is associated with a poor prognosis in lung adenocarcinoma. This study assessed the prognostic impact of the preoperative peripheral monocyte count in lung adenocarcinoma. Materials and methods: We retrospectively analyzed 302 consecutive patients with lung adenocarcinoma who received curative resection at Kitano Hospital. The receiver operating characteristic (ROC) curve for the peripheral monocyte count was used to determine the cut-off value. The relations between peripheral monocyte counts and clinicopathological factors were assessed. We also evaluated the impacts of possible prognostic factors including the preoperative peripheral monocyte count on survival, using the two-tailed log-rank test and Cox proportional hazards model. In addition, immunohistochemical staining for CD68 was performed to evaluate the monocytes in primary tumors. Results: A peripheral monocyte count of 430 mm−3 was the optimal cut-off value for prognosis. An elevated peripheral monocyte count was significantly associated with sex, performance status, smoking history, chronic obstructive pulmonary disease and interstitial lung disease. The two-tailed log-rank test demonstrated that patients with an elevated peripheral monocyte count experienced a poorer recurrence-free survival (RFS) and overall survival (OS) (P = 0.0063, P < 0.0001, respectively). In the multivariate analysis an elevated peripheral monocyte count was shown to be an independent prognostic factor for the RFS and OS (HR: 1.765; 95% CI: 1.071–2.910; P = 0.0258, HR: 4.339; 95% CI: 2.032–9.263; P = 0.0001, respectively). Furthermore, numbers of the monocytes in primary tumors significantly correlated with peripheral monocyte counts (r = 0.627, P < 0.0001). Conclusion: The preoperative peripheral monocyte count is an important prognostic factor for patients with lung adenocarcinoma after curative resection. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is one of the most common causes of cancerrelated death and adenocarcinoma is the most popular type of
∗ Corresponding author at: Respiratory Disease Center, Tazuke Kofukai Medical Research Institute, Kitano Hospital, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan. Tel.: +81 6 6312 8831; fax: +81 6 6361 8867. E-mail address: [email protected] (C.-l. Huang). http://dx.doi.org/10.1016/j.lungcan.2014.06.015 0169-5002/© 2014 Elsevier Ireland Ltd. All rights reserved.
lung cancer, accounting for approximately one-half of lung cancer cases. In lung adenocarcinoma, tumor-associated macrophages (TAMs) were reported to be associated with a poor prognosis [1,2]. TAMs are derived from monocytes and myeloid progenitor cells [3]. An elevated peripheral monocyte count at presentation has recently been shown to predict poor prognosis in various types of malignancy, including malignant lymphomas [4–7]. However, it is unknown if an elevated peripheral monocyte count is associated with a poor prognosis in lung adenocarcinoma. This study assessed the prognostic impact of the preoperative peripheral monocyte
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count on the recurrence-free survival (RFS) and overall survival (OS) in patients with lung adenocarcinoma who received curative resection. 2. Materials and methods
compared using the chi-square test. Continuous variables were compared using the Wilcoxon rank-sum test. All statistical analyses were performed using the statistical software R version 2.13.1 (R Foundation for statistical computing, Vienna, Austria). All P values are 2-sided, and P values less than 0.05 were considered statistically significant.
2.1. Patients We conducted a retrospective analysis of patients diagnosed with lung adenocarcinoma who underwent surgery at the Tazuke Kofukai Medical Research Institute, Kitano Hospital between January 2007 and December 2012. All patients met the following criteria: pathological confirmation of lung adenocarcinoma; complete curative resection; no preoperative treatment; no microscopic residual tumor; no history of transplantation, immunosuppression; no evidence of infection such as pneumonia before surgery; no treatment for concomitant autoimmune diseases with immunosuppressive therapy; no history of acute exacerbation of chronic obstructive pulmonary disease (COPD) or interstitial lung disease (ILD) within a month before surgery; negative for anti-HIV; and availability of laboratory data and follow-up information. COPD was diagnosed on the basis of the Global Initiative for Chronic Obstructive Lung Disease [8]. ILD was diagnosed by two pulmologists, based on medical history, physical examination and abnormalities compatible with bilateral lung fibrosis on high-resolution computed tomography. A peripheral venous blood sample was collected from each patient within a month before surgery. A blood test was performed using a fully automated blood cell counting system. Histological classification was carried out according to the WHO guidelines [9]. Disease stages were based on the 7th edition of TNM classification of malignant tumors [10]. Study approval was granted by the ethical committee of the Tazuke Kofukai Medical Research Institute, Kitano Hospital, in accordance with the Declaration of Helsinki. 2.2. Evaluation of clinicopathological factors The following clinical characteristics were retrieved from the available clinical records: age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, smoking history, pathological stage, pathological tumor status, pathological lymph node status, pleural invasion, vascular invasion, lymphatic permeation, peripheral leukocyte counts, peripheral monocyte counts, peripheral neutrophil counts, peripheral lymphocyte counts, and C-reactive protein (CRP) levels. RFS was measured from the date of surgery until the date of recurrence or until the date the patient was last known to be disease free. OS was measured from the date of surgery until the date of death from any cause or until the date on which the patient was last known to be alive. 2.3. Statistical analysis We estimated OS and RFS using the Kaplan–Meier analysis [11]. Differences between survival curves were tested for statistical significance using the two-tailed log-rank test. Univariate and multivariate prognostic analyses were performed for OS and RFS outcomes using the Cox proportional hazards model. For multivariate survival analysis, a stepwise backward procedure to derive a final model of the variables that had a significant independent relationship with survival was employed. To remove a variable from the model, the corresponding P value had to be >0.10. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off values for peripheral leukocyte counts, peripheral neutrophil counts, peripheral monocyte counts, and peripheral lymphocyte counts; values with maximum joint sensitivity and specificity were selected. Categorical variables were
2.4. Immunohistochemistry Formalin-fixed paraffin-embedded tissue was cut into 4m sections and mounted on poly-l-lysine-coated slides. After deparaffinization and rehydration, the slides were heated in a microwave for 30 min in a 10-mol/L citrate buffer solution at pH 6.0, and were cooled to room temperature for 20 min. After quenching the endogenous peroxidase activity with 0.3% H2 O2 (in absolute methanol) for 30 min, the sections were treated with 5% bovine serum albumin to block non-specific staining. Individual sections were incubated overnight with mouse monoclonal antihuman CD68 antibody (Clone KP1, Dako, Japan) at a 1:100 dilution. The slides were then incubated with EnVision (Dako, Denmark) for 1 h at room temperature. Then, the antibody binding was visualized with 3,3 -diaminobenzidine tetrahydrochloride. Finally, the sections were lightly counterstained with Mayer’s hematoxylin. All of the immunostained sections were reviewed by two authors (S.K. and C.H.) without knowledge of the patients’ characteristics. CD68positive round cells in the stroma of the cancer tissue were counted as macrophage. Cases with discrepancies were jointly reevaluated until a consensus was reached. 3. Results 3.1. Patients Data from 445 patients diagnosed with non-small cell lung cancer (NSCLC) who underwent surgery at our hospital between January 2007 and December 2012 were obtained from the hospital’s database. One hundred and forty-three patients were excluded due to diagnosis of histology other than lung adenocarcinoma (N = 107), preoperative treatment (N = 14), and incomplete resection (N = 22). Thus 302 patients were included in this study. The clinicopathological characteristics of patients are shown in Table 1. There were 136 men and 166 women. The median age at the time of surgery was 67 years (range: 34–87 years). The median follow-up duration was 33.4 months (range: 1.0–77.4 months). Fifty-seven patients received postoperative adjuvant chemotherapy and 72 patients experienced recurrences of lung adenocarcinoma. The 5-year recurrence-free survival rate and overall survival rate of all 302 patients were 70.2% (pathological stage I, 83.7%; pathological stage II, 43.4%; pathological stage IIIA, 34.9%) and 84.0% (pathological stage I, 87.5%; pathological stage II, 73.7%; pathological stage IIIA, 75.5%), respectively. 3.2. Optimal cut-off values for leukocyte, neutrophil, monocyte and lymphocyte counts ROC curves for peripheral leukocyte counts, peripheral neutrophil counts, peripheral monocyte counts and peripheral lymphocyte counts were used to determine cut-off values. The area under the curve (AUC) for peripheral leukocyte counts was 0.560 (95% confidence interval (95% CI), 0.443–0.677). A leukocyte count of 5400 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (88.9% sensitivity and 47.0% specificity). The AUC for peripheral neutrophil counts was 0.606 (95% CI: 0.467–0.744). A neutrophil count of 2900 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve
S. Kumagai et al. / Lung Cancer 85 (2014) 457–464 Table 1 Clinicopathological characteristics of patients. Variables
Total (N = 302)
Age (median), years Sex (male/female) ECOG performance status (0/1/2) Smoking history (never/ever) Comorbidity and past history COPD ILD Old pulmonary tuberculosis Bronchial asthma Hypertension Diabetes mellitus Hyperlipidemia Ischemic heart disease Arrhythmia Resected side (right/left) Surgical procedure (Lob/Seg/Wedge) Pathological stage (I/II/IIIA) Tumor status (T1/T2/T3/T4) Lymph node status (N0/N1/N2) Pleural invasion (present/absent) Vascular invasion (present/absent) Lymphatic permeation (present/absent) Leukocyte counts (mean ± SD), mm−3 Neutrophil counts (mean ± SD), mm−3 Monocyte counts (mean ± SD), mm−3 Lymphocyte counts (mean ± SD), mm−3 C-reactive protein (mean ± SD), mg/dL Postoperative adjuvant chemotherapy Recurrences of lung adenocarcinoma Cause of death (lung cancer/others/unknown)
67 (34–87) 136/166 271/30/1 159/143 38 22 15 22 84 29 40 19 20 180/122 259/39/4 220/39/43 173/97/20/12 254/14/34 71/231 41/260 23/276 5900 ± 1700 3600 ± 1600 410 ± 150 1700 ± 560 0.28 ± 0.52 57 72 15/16/4
Abbreviations: SD, standard deviation; ECOG, Eastern Cooperative Oncology Group; COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease; Lob, lobectomy or bilobectomy; Seg, segmentectomy; Wedge, wedge resection.
(100.0% sensitivity and 42.6% specificity). The AUC for peripheral monocyte counts was 0.711 (95% confidence interval (CI), 0.622–0.800) (Suppl. 1). A peripheral monocyte count of 430 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (69.7% sensitivity and 66.9% specificity). The AUC for peripheral lymphocyte counts was 0.663 (95% CI: 0.444–0.882). A lymphocyte count of 1400 mm−3 corresponded to the maximum joint sensitivity and specificity on the ROC curve (66.7% sensitivity and 70.4% specificity). Supplementary Fig. 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan.2014.06.015. 3.3. The relations between peripheral monocyte counts and clinicopathological factors The relations between the peripheral monocyte count and clinicopathological factors are shown in Table 2. An elevated peripheral monocyte count was significantly associated with sex, ECOG performance status, smoking history, COPD, and ILD. An elevated peripheral monocyte count was also significantly associated with recurrences of lung adenocarcinoma. 3.4. Evaluation of the prognostic impact of peripheral monocyte counts The Kaplan–Meier analysis was performed to determine if the peripheral monocyte count was associated with the RFS and OS. The RFS and OS were significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 , in all stages (P = 0.0063 and P < 0.0001, respectively) (Fig. 1A and B). In the pathological stage I patients, the OS and RFS were significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the
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group with a peripheral monocyte count ≤430 mm−3 (P = 0.0050 and P = 0.0149, respectively) (Fig. 1C and D). In the pathological stage II patients, the RFS was not significantly shorter in the group with a peripheral monocyte count >430 mm−3 (P = 0.0549) (Fig. 1E). However, the OS was significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 (P = 0.0099) (Fig. 1F). In the pathological stage IIIA patients, there was no significant difference in RFS between the two groups (P = 0.1740) (Fig. 1G). In contrast, the OS was significantly shorter in the group with a peripheral monocyte count >430 mm−3 compared to the group with a peripheral monocyte count ≤430 mm−3 (P = 0.0398) (Fig. 1H). 3.5. Univariate and multivariate analyses of factors associated with prognosis As for the RFS, the univariate analysis identified eleven significant risk factors: sex, ECOG performance status, tumor status, lymph node status, pleural invasion, vascular invasion, lymphatic permeation, a high peripheral monocyte counts, a low peripheral lymphocyte count, a high CRP level, and ILD (Table 3). In the multivariate analysis, a high peripheral monocyte count was shown to be a statistically significant independent predictor of the RFS (HR: 1.765; 95% CI: 1.071–2.910; P = 0.0258). The other independent prognostic factors were sex (HR: 1.689; 95% CI: 1.023–2.789, P = 0.0406), tumor status (HR: 2.472; 95% CI: 1.458–4.193, P = 0.0008), lymph node status (HR: 3.400; 95% CI: 2.044–5.657, P < 0.0001), and a low lymphocyte count (HR: 2.128; 95% CI: 1.319–3.433, P = 0.0020), and ILD (HR: 2.093, 95% CI: 1.063–4.121, P = 0.0327). The univariate analysis showed ten significant risk factors for the OS: age, sex, ECOG performance status, tumor status, a high peripheral neutrophil count, a high peripheral monocyte count, a low peripheral lymphocyte count, a high CRP level, COPD, and ILD (Table 4). The multivariate analysis revealed that a high peripheral monocyte count was a statistically significant independent prognostic factor of the OS (hazard ratio (HR): 4.339; 95% CI: 2.032–9.263; P = 0.0001). The other independent prognostic factors were tumor status (HR: 2.131; CI: 1.026–4.425, P = 0.0425), a low peripheral lymphocyte count (HR: 3.365; 95% CI: 1.683–6.725, P = 0.0006), and ILD (HR: 3.041; 95% CI: 1.337–6.914, P = 0.0080). The peripheral monocyte count was analyzed in an attempt to further discriminate clinical outcomes in lung adenocarcinoma by other prognostic factors: pathological stage; tumor status; lymph node status; peripheral leukocyte counts; peripheral neutrophil counts; peripheral lymphocyte counts; CRP. Interactions of a peripheral monocyte count of >430 mm−3 vs. a peripheral monocyte count of ≤430 mm−3 were shown in a Forest plot (Fig. 2). Patients with a peripheral monocyte count of >430 mm−3 showed a poorer OS, compared to those with a peripheral monocyte count of ≤430 mm−3 in 13 of 14 subgroups, the exception being the group of patients with T1. 3.6. Correlations between numbers of monocytes in primary tumors and peripheral monocyte counts In order to evaluate the monocytes in the stroma of the primary tumor, immunohistochemistry was performed using consecutive 71 patients who underwent surgery at our hospital between June 2011 and December 2012 (Suppl. 2). The mean number of CD68-positive monocytes in the stroma of primary tumors was 29.7 ± 29.9 (median: 20, range: 0–122). The numbers of monocytes in the stroma of primary tumors significantly correlated
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Fig. 1. The Kaplan–Meier analyses of recurrence-free survival and overall survival stratified by peripheral monocyte counts (<430 vs. ≥430 mm−3 ) are shown for all pathological stages (A, B, respectively), for pathological stage I (C, D, respectively), for pathological stage II (E, F, respectively), and for pathological stage IIIA (G, H, respectively).
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Table 2 Relationship between preoperative peripheral monocyte counts and clinicopathological factors. Monocyte counts
Variables
Age (median), years Sex (male/female) ECOG performance status (0/1/2) Smoking history (never/ever) Comorbidity and past history COPD ILD Old pulmonary tuberculosis Bronchial asthma Hypertension Diabetes mellitus Hyperlipidemia Ischemic heart disease Arrhythmia Resected side (right/left) Surgical procedure (Lob/Seg/Wedge) Pathological stage (I/II/IIIA) Tumor status (T1/T2/T3/T4) Lymph node status (N0/N1/N2) Pleural invasion (present/absent) Vascular invasion (present/absent) Lymphatic permeation (present/absent) Postoperative adjuvant chemotherapy Recurrences of lung adenocarcinoma Cause of death (lung cancer/others/unknown)
P-value
≤430 mm−3 N = 192
>430 mm−3 N = 110
68 (34–87) 66/126 179/13/0 120/72
68.5 (45–87) 70/40 92/17/1 39/71
0.7977 <0.0001 0.0115 <0.0001
13 8 9 14 48 18 25 11 11 114/78 169/21/2 139/27/26 110/60/15/7 163/8/21 42/150 23/169 13/179 37 36 4/6/1
24 14 6 8 36 11 15 8 9 66/44 90/18/2 81/12/17 63/37/5/5 91/6/13 29/81 18/92 10/100 20 36 11/10/3
0.0002 0.0099 0.7870 0.9999 0.1820 0.8420 0.8620 0.6270 0.4730 0.9999 0.3420 0.7050 0.7130 0.8190 0.3990 0.2990 0.5030 0.8790 0.0076 0.8740
Abbreviations: SD, standard deviation; ECOG, Eastern Cooperative Oncology Group; COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease; Lob, lobectomy or bilobectomy; Seg, segmentectomy; Wedge, wedge resection.
with peripheral monocyte counts (r = 0.627, P < 0.0001). The median number of monocytes was used to divide the 71 cases into two groups: monocyte-high; above the median value, monocyte-low; below the median value. The peripheral monocyte counts were 470 ± 146 mm−3 in monocyte-high tumors, and 328 ± 117 mm−3 in monocyte-low tumors. The peripheral monocyte counts were significantly higher in monocyte-high tumors than in monocyte-low tumors (P < 0.0001).
Supplementary Fig. 2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan.2014.06.015. 4. Discussion We demonstrated that a high peripheral monocyte count was a poor prognostic factor in lung adenocarcinoma. Monocytosis at presentation was reported to predict a poor prognosis
Table 3 Prognostic impacts on recurrence-free survival in lung adenocarcinoma. Variables Univariate analysis Age Sex ECOG performance status Smoking history Tumor status Lymph node status Pleural invasion Vascular invasion Lymphatic permeation Leukocyte counts Monocyte counts Neutrophil counts Lymphocyte counts CRP COPD ILD Multivariate analysis Sex Tumor status Lymph node status Monocyte counts Lymphocyte counts ILD
HR
95% CI
P-value
≥70 Male 1/2 Ever T2/T3/T4 N1/N2 Present Present Present >5400 mm−3 >430 mm−3 >2900 mm−3 ≤1400 mm−3 >0.1 mg/dL Present Present
1.499 1.876 1.924 0.652 3.502 5.081 3.370 3.353 4.332 1.377 1.886 1.436 1.762 1.885 1.616 3.507
0.911–2.304 1.175–2.995 1.009–3.668 0.409–1.038 2.130–5.756 3.156–8.178 2.120–5.356 1.909–5.887 2.336–8.033 0.858–2.211 1.187–2.995 0.879–2.346 1.108–2.803 1.186–2.995 0.868–3.010 1.878–6.547
0.1174 0.0084 0.0469 0.0716 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.1850 0.0072 0.1489 0.0168 0.0073 0.1301 <0.0001
Male T2/T3/T4 N1/N2 >430 mm−3 ≤1400 mm−3 Present
1.689 2.472 3.400 1.765 2.128 2.093
1.023–2.789 1.458–4.193 2.044–5.657 1.071–2.910 1.319–3.433 1.063–4.121
0.0406 0.0008 <0.0001 0.0258 0.0020 0.0327
Abbreviations: ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; 95% CI, 95% confidence interval; CRP, C-reactive protein; COPD, chronic obstructive lung disease; ILD, interstitial lung disease.
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Table 4 Prognostic impacts on overall survival in lung adenocarcinoma. Variables Univariate analysis Age Sex ECOG performance status Smoking history Tumor status Lymph node status Pleural invasion Vascular invasion Lymphatic permeation Leukocyte counts Neutrophil counts Monocyte counts Lymphocyte counts CRP COPD ILD Multivariate analysis Tumor status Monocyte counts Lymphocyte counts ILD
HR
95% CI
P-value
≥70 Male 1/2 Ever T2/T3/T4 N1/N2 Present Present Present >5400 mm−3 >2900 mm−3 >430 mm−3 ≤1400 mm−3 >0.1 mg/dL Present Present
2.856 2.471 2.610 0.607 2.428 1.913 1.717 2.072 1.376 1.483 2.337 3.848 2.429 2.166 2.368 5.717
1.436–5.677 1.227–4.976 1.131–6.024 0.311–1.187 1.217–4.842 0.895–4.092 0.859–3.430 0.850–5.053 0.420–4.507 0.743–2.959 1.059–5.159 1.884–7.858 1.246–4.737 1.107–4.237 1.068–5.252 2.662–12.28
0.0028 0.0113 0.0246 0.1447 0.0118 0.0943 0.1261 0.1093 0.5978 0.2633 0.0356 0.0002 0.0092 0.0240 0.0339 <0.0001
T2/T3/T4 >430 mm−3 ≤1400 mm−3 Present
2.131 4.339 3.365 3.041
1.026–4.425 2.032–9.263 1.683–6.725 1.337–6.914
0.0425 0.0001 0.0006 0.0080
Abbreviations: ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; 95% CI, 95% confidence interval; CRP, C-reactive protein; COPD, Chronic obstructive lung disease; ILD, interstitial lung disease.
in classical Hodgkin lymphoma [4], nodular lymphocytepredominant Hodgkin lymphoma [5], follicular lymphoma [6], and mantle cell lymphoma [7]. To the best of our knowledge, this is the first report showing the prognostic significance of the peripheral monocyte count in lung adenocarcinoma, one of the most popular solid tumors.
Peripheral monocytes and myeloid progenitor cells grow into TAMs when entering tumors [3]. TAMs are classified into the two phenotypes: the classically activated type 1 macrophages (M1) and the alternatively activated type 2 macrophages (M2) [12]. Macrophages are polarized along the M1 pathway, affected by bacterial moieties such as lipopolysaccharide (LPS), Th1
Fig. 2. Subgroup analysis of overall survival for peripheral monocyte counts (<430 vs. ≥430/mm−3 ) is shown in the Forest plot.
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cytokine interferon-gamma (IFN␥). Activated M1 macrophages, which are adapted to promote an anti-tumor response, eliminate tumor cells, present antigen to T cells for an adaptive immune response, and produce cytokines [12,13]. In contrast, exposure to Th2 and tumor-derived cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), transforming growth factor beta (TGF-) or prostaglandin E2 (PGE2) promote M2 polarization [14]. M2 macrophages, suitable for tumor development, express high levels of class A scavenger receptor (CD204) and mannose receptor (CD163) [15,16], suppress Th1 mediated-inflammation through IL-10 and IL-1b production, and promote angiogenesis via vascular endothelial growth factor (VEGF) production [17–19]. It was shown that the infiltration of macrophages in tumor islets was associated with a good prognosis in contrast to infiltration in the tumor stroma, related to a worse prognosis in NSCLC [20,21]. These findings suggest that the localization of TAMs plays an important role in predicting prognosis. Recent studies have shown that macrophages in the tumor islets which are associated with a good prognosis are mainly of the M1 phenotype [22,23]. On the other hand, TAMs of the M2 phenotype, which are located in the tumor stroma, were correlated with lymph node metastasis and poor prognosis in lung adenocarcinoma [1,2,24]. In classical Hodgkin lymphoma, the correlation between the percentage of peripheral monocytes at diagnosis and number of CD163+ TAMs in lesional tissue was reported [25]. In the present study, an elevated peripheral monocyte count is significantly associated with a poor prognosis in lung adenocarcinoma. Furthermore, numbers of monocytes in primary tumors significantly correlated with peripheral monocyte counts. A recent study has also reported that circulating macrophages predict postoperative recurrence in non-small-cell lung cancer patients [26]. These results may suggest a complex relation between peripheral monocytes and TAMs of the M2 immunosuppressive phenotype in lung adenocarcinoma. In this study, an elevated peripheral monocyte count was significantly associated with sex, ECOG performance status, smoking history, COPD and ILD. Cigarette smoking was reported to increase the peripheral monocyte count [27]. Systemic inflammation is associated with impaired lung function, particularly among those with extensive cigarette smoking [28]. The proportion of smokers in men is known to be larger than that in women. In addition, COPD was shown to be associated with lung cancer mortality [29]. On the other hand, in patients with ILD the peripheral monocyte count is elevated [30]. Recently, Voltolini et al. reported that long-term survival of patients with NSCLC and ILD is significantly lower when compared with patients without ILD [31]. In the present study, the univariate analysis showed that both COPD and ILD were significantly associated with the OS of lung adenocarcinoma. Besides, the multivariate analysis demonstrated that ILD was a significant prognostic factor of the OS and RFS, and that the peripheral monocyte count predicted the prognosis of the OS and RFS, independent of ILD. We also evaluated the prognostic importance of the peripheral monocyte count in patients without COPD or ILD (N = 251). The univariate analysis demonstrated that a high peripheral monocyte count was a statistically significant predictor of the OS (P = 0.0087). The result reinforced the hypothesis that the peripheral monocyte count is associated with the prognosis of lung adenocarcinoma, independent of COPD and ILD. Both peripheral neutrophil counts [32,33] and CRP levels [34] have been shown to be independent prognostic factors in NSCLC. In this study, however, we showed that an elevated peripheral neutrophil count and CRP level were poor prognostic factors only in the univariate analysis, not in the multivariate analyses. Both peripheral neutrophil counts and CRP levels were supposed to reflect
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the nature and severity of inflammation associated with tumor. In contrast, an elevated peripheral monocyte count was shown to predict a poor RFS and OS in the multivariate analysis, as well as in the univariate analysis. In addition, as shown in the Forest plot, the peripheral monocyte count was found to predict the prognosis of lung adenocarcinoma in 13 subgroups. These findings may suggest that peripheral monocyte counts is a very strong prognostic factor and play a significant role in the tumor progression. The preoperative peripheral lymphocyte count was reported to be an important prognostic factor in node-negative NSCLC [35]. The present study revealed that the peripheral lymphocyte count was a prognostic factor in patients with lung adenocarcinoma including node-positive carcinoma that received complete resection. In addition, the multivariate analysis indicated that a low peripheral lymphocyte count predicted a poor RFS and OS, independent of other possible prognostic factors. In previous study, the cut-off values of the peripheral lymphocyte count differed from the cut-off value found in the present study, possibly reflecting the difference in the type of histology. Fifty-seven patients received postoperative adjuvant chemotherapy in this study. We evaluated the impact of postoperative adjuvant chemotherapy on the OS. However, there was no difference in the OS between patients with postoperative adjuvant chemotherapy and those without by the two-tail log-rank test (P = 0.2330). Among 245 patients without postoperative adjuvant chemotherapy, the two-tail log-rank test showed that peripheral monocyte count >430 mm−3 was also a significant prognostic factor for the OS in all pathological stages (N = 245), pathological stage I (N = 213), and pathological stage II–IIIA (N = 32) (OS, P = 0.0008, P = 0.0252, P = 0.0057, respectively; RFS, P = 0.0003, P = 0.0039, P = 0.0018, respectively). A peripheral monocyte count >430 mm−3 was shown to be associated with a poor prognosis, even negating the effects of postoperative adjuvant chemotherapy. In the present study, twenty patients (57.1% of all cases of death) have died of causes unrelated to lung adenocarcinoma. Therefore, we also assessed the cancer-specific survival (CSS), which was measured from the date of surgery until the date of death from lung adenocarcinoma or its treatment, with data for patients who died of unrelated causes censored at the time of death or last follow-up. The univariate analysis demonstrated that a peripheral monocyte count >430 mm−3 was a significant predictor of a poor CSS (P = 0.0074). This result may support the hypothesis that peripheral monocytes are significantly associated with the progression of lung adenocarcinoma. In conclusion, our study demonstrated that preoperative peripheral monocyte count is a useful prognostic marker in lung adenocarcinoma, supporting the notion that the TAMs have a prognostic impact on clinical outcome in lung adenocarcinoma. The peripheral monocyte count, obtained from a CBC before surgery, may be one of the simplest measures as a prognostic marker in lung adenocarcinoma. Limitations of this study include retrospective analysis, relatively short follow-up duration. Further prospective studies are required to confirm the present findings and to clarify the mechanism in which peripheral monocytes have impacts on clinical outcomes.
Conflict of interest statement All authors have no potential conflicts of interest to report.
Acknowledgements No financial support for this study was provided.
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